Unit 4 Bacteria

7/28/2019 Unit 4 Bacteria

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BACTERIAL CELLObjectives:

1.State the shapes and arrangements of named examples of

bacteria

2.Describe the appendages and inclusions found in variousexamples of bacteria

3.Distinguish between simple, differential and special stains

4.State the differences between the cell walls of Gram positiveand Gram negative bacteria and how these relate to stainingcharacteristics.

5.Describe the bacterial endospore and explain its role anddescribe its role in the survival of the bacteria.


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BACTERIAL CELLBACTERIAL CELL

Bacteria come in many shapes.

most are spherical (called cocci, singularCoccus),

or rod shaped (called bacilli, singular. bacillus

and all enteric bacteria eg. E. coli, Clostridiums .

or spiral-shaped (called spirilla, singular.Spirillium-Treponema palladium, Leptospira

interrogans). There are some intermediate shapes. The most

common are short bacillus (calledcoccobacillus), and a short comma shaped

spirillium (eg. vibrio-vibrio cholerae).


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BACTERIAL MORPHOLOGYBACTERIAL MORPHOLOGY

CoccusSpirochete

Rod

Spirillium

Budding and appendage

Filamentous


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The Cocci or round

A. Division in one plane produces one of twoarrangements:

1. diplococci: cocci arranged in pairs(genera Neisseria eg. N. meningitidis and N.gonorrhoeae)


2. streptococci: cocci arranged in chains(genera Streptococcus eg. S. pneumoniae, S.mutans,S. Pyogenes)

B. Division in two planes produces squares of 4called tetrads arrangement.

(genera micrococcus eg. M. luteus and M. roseus)


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C. Division in three planes produces asarcina arrangement.

sarcinae: cocci in arranged cubes of 8(Genera Sarcina eg. S. ventriculi and S.

lutea)


D. Division in random planes produces a

irregular, often grape-like clusterscalled Staphylococci.

(Genera staphylococcus: eg S. aureus andS. epidermidis)


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BACTERIAL MORPHOLOGYBACTERIAL MORPHOLOGYBACTERIAL MORPHOLOGYBACTERIAL MORPHOLOGY


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Bacilli or rod shaped

Bacilli all divide in one plane producing abacillus, diplobacillus, streptobacillus,palisades or coccobacillus arrangement.


b) streptobacillus: bacilli arranged in chains

c) Palisades: vertical side by side

d) coccobacillus: oval and similar to a coccus(eg. Haemophilus influenzae and Chlamydia

trachomatis )


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OTHER BACTERIAL CELL STRUCTURESOTHER BACTERIAL CELL STRUCTURES


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Spiral

a)vibrio: a curved or comma-shaped rod(eg. V. cholerae and V. parahaemolyticus)

b)spirillum: a thick, rigid spiral

(eg. Spirillum minus)


c)spirochete: a thin, flexible spiral (eg. Treponema

pallidum and Borrelia recurrentis)

Spiral bacteria usually remain as single microorganisms however they vary in the number ofcorkscrew turns.


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BA

CTERIALM

BA

CTERIALMORPH

OLOGY

ORPH

OLOGY


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Exceptions to the above shapes

Sheathed (actinomyces, Sphaerotilus)

Stalked (Gallionella ferruginea)

Filamentous (Herbidospora cretacea)


Square (Walsbys square bacterium)

Star-shaped

Spindle-shaped

Lobed, and (hyphomicrobium)

Pleomorphic


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BACTERIAL CELL STRUCTURESBACTERIAL CELL STRUCTURES

CAPSULE

Pil iF lagel lum


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PILLI

Straight hair-like appendages which tend

to be short.

They are made of the protein pillin whichis arranged helically around a central hollow

core.

Pilli function to attach bacterial cells toother cells.

The protein called adhesions found in eitherthe tip or side of pilli make the connectionpossible.


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Sex pilli attach one bacterial cell toanother during mating.

While others attach them to plant or

PILLI

in a favorable environment.

Escher ich ia col iand Neisser iagono r rhoeaeboth have pilli.


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FLAGELLUM

The function of the flagella is LOCOMOTION

Flagella structure has three distinct parts:

1. An outer helical-shaped filament-

Composed of subunits of the protein flagellinarranged in a helical manner around a hollow

core.

2. A hook- the filament is attached to a hookwhich allows the filament to move in differentdirections. The hook is attached to the basalbody.


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3. A basal body- Anchors the flagella tothe envelope and causes it to rotate.

The part of the basal body that

penetrates the envelope has four (in

FLAGELLUM

gram nega ve or ree n grampositive) rings.

In gram negatives the L ring isembedded in the outer membranehowever Gram-positives lack this

ring.


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The other rings are the P (peptidoglycan),and the inner S(superficial) and M(membrane) rings.

The motor that rotates the flagellum is a

FLAGELLUM

cytoplasm.

The core of the flagellum (the rod)rotates inside the rings which act asanchors to the envelope.


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FLAGELLUM


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Different types of bacteria have different

numbers of flagella: Monotrichous (generapseudomonas), amphitrichous, lophotrichousand peritrichous (genera Escherichia).

A group of bacteria called the spirocheteshave flagella which are bundled into two axialfilaments which are trapped inside theperiplasm.


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OTHER BACTERIAL CELL STRUCTURESOTHER BACTERIAL CELL STRUCTURES

Different types of bacteria have differentnumbers of flagella:

Monotrichouseg (pseudomonas)

Amphitrichous

Peritrichouseg Escherichia


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CAPSULE

Most bacteria secrete a slimy orgummy substance that formsoutermost layer of the cell.

Capsules vary in thickness andcomposition with the organism that

produces it.

Most are however made ofpolysaccharide and a few of

protein.


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CAPSULECAPSULE


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Functions of the Capsule:

1. Principally protect the cell against dryingout

2. Adhere cells to a surface where

CAPSULE

3. Protect disease causing bacteriaagainst phagocytosis thus play animportant role in infection.

Stains ofS. pn eum on iaethat lack a capsuleare harmless because they are quickly

consumed.


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SHEATH

Some bacteria develop within sheaths which are long

transparent polysaccharide tubes.

Cells divide and grow inside the tubeelongating the tube to fit the cells.

These organisms have a free-swimming stagewhere they exit sheath and move by flagella

Sheathed bacteria are found in contaminated streams

and sewage treatment ponds. Sheath serves as protection against debris

Allows survival over a wide pH range


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SHEATHED BACTERIA


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NUCLEOID

The nucleoid or nuclear region is welldefined even though it is not membranebound.

It is a mass of DNA-carries the cells geneticinformation.

Bacterial DNA (chromosomal) is usuallyarranged in a single circular molecule.

Usually they also contain smaller circularDNA molecules called plasmids.


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RIBOSOMES

Found in the cytoplasm.

Their great number and small size give thecytoplasm its characteristic grainy appearance.

The ribosome is the site of protein

synt es s

A ribosome is composed of two subunits

both composed of protein and RNA. The large subunit of prokaryotic cells is smaller

than that of Eukaryotic cells (80s).


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Two complexes of RNA and protein makeup the prokaryotic ribosome, the 30Ssubunit and the 50S subunit.

The 30S subunit is composed of 21 proteins and a-

RIBOSOMES

,nucleotides, termed the 16S rRNA.

The 50S subunit contains 31 proteins and two RNAspecies, a 5S rRNA of 150 nucleotides and a 23SrRNA of about 2,900 nucleotides.


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BASIC STRUCTURE OF BACTERIABASIC STRUCTURE OF BACTERIA

Ribosome


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STORAGE GRANULES

Many bacterial species have several kinds of

storage granules.

Granules of carbon containing compounds like

glycogen and poly-beta-hydroxyalkanes.

Other granules containing reserves ofsulphur and nitrogen, and granules of

polyphosphate.


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OTHER INCLUSIONS

Gas vacuoles: gas-filled regions surroundedby a monolayer of a single protein that allowsthe bacteria to float at the water level with theconditions best suited for photosynthesis.

Ch lo r osom es: een n p o osyn e cbacteria. These structures house accessorypigments necessary for photosynthesis.


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Magn et osom es:

These magnet-like structures are neededfor magnetotaxis.

OTHER INCLUSIONS

They allow the bacteria to follow magneticlines of force toward the bottom of bodies

of water- their optimum environment.


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Hete rocys t s:

Nitrogen fixation and oxygenic photosynthesis areincompatible since nitrogen fixing systems areextremely sensitive to oxygen.

Many cyanobacteria solve this problem by carrying

out nitrogen-fixation in specialized cells called

heterocysts.

All other cells photosynthesize.


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STAINS

Unstained bacteria are practicallytransparent when viewed using the lightmicroscope and thus are difficult to see.

Stains serve several purposes:

. ta ns eren a e m croorgan sms romtheir surrounding environment

2. They allow detailed observation of microbial

structures at high magnification3. Certain staining protocols can help to

differentiate between different types ofmicroorganisms.


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3 basic types: simple, differential, &specialized.

Simple stains react uniformly with allmicroorganisms and only distinguish the organismsfrom their surroundings. Differentiation of cell

types or structures is not the objective of the

STAINS

.

However, certain structures which are not stained by thismethod may be easily seen, for example, endospores andlipid inclusions

Differential stains discriminate between variousbacteria, depending upon the chemical or physical

composition of the microorganism.


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Types of dyes and stains usedTypes of dyes and stains used

SimpleStains

DifferentialStains

StructuralStains

STAINING BACTERIASTAINING BACTERIA

Basic Dyescationic + charged

Gram Stain Endospore Stain

Acidic Dyesanionic, - charged Acid-fast stain Capsule Stain

Indifferent Dyes Flagella Stain


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ACID FAST STAIN

Because of the waxy mycolic acids present on

the cell walls, cells of species ofMycobacterium do not stain readily withordinary dyes.

However, treatment with cold carbol fuchsinfor several hours or at high temp for 5 mins

will dye the cells.

Once the cells have been stained, subsequenttreatment with a dilute hydrochloric acidsolution or ethyl alcohol containing 3% HCl

(acid-alcohol) will not decolorize them.


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ACID FAST STAIN

Such cells are thus termedacid-fast in that the cell willhold the stain fast in thepresence of the acidic

.

This property is possessedby few bacteria other thanMycobacterium.


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STRUCTURAL STAINS

Specialized stains detect specific structures of

cells such as flagella and endospores

Endospore Stain

vigorous treatment for staining, but oncestained, the endospores are difficult to

decolorize. In the endospore stain, water is the

decolorizing agent that removes the primary

stain from the vegetative cells.


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STRUCTURAL STAINS

Endospore Stain

Endospore stains require heat to drivethe stain into the cells.

For an endospore stain to be successful,

boiling and the stain cannot dry out.

Most failed endospore stains occurbecause the stain was allowed tocompletely evaporate during theprocedure.


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Named after the Danish physician,

Christian Gram, who developed thisstaining technique in 1884.

1. Bacterial cells are dried onto a glass slide

GRAMS STAIN

an s a ne w crys a v o e , en was ebriefly in water.

2. Iodine solution (mordant) is added so thatthe iodine forms a complex with crystalviolet in the cells.


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3. Alcohol or acetone is added to solubilise

the crystal violet - iodine complex.

4. The cells are counterstained with safranin,

then rinsed and dried for microscopy.

GRAMS STAIN

Gram-positive cells retain the crystal violet-iodine complex and thus appear purple.

The thickness of this wall blocks the escape ofthe crystal violet-iodine complex when the cellsare washed with alcohol or acetone.


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Gram-negative bacteria have only a thin

layer of peptidoglycan, surrounded by athin outer membrane composed oflipopolysaccharide (LPS).

GRAMS STAIN

r wthe peptidoglycanand LPS layers is

termed thePERIPLASMICSPACE.


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The PERIPLASMIC SPACE is a fluid orgel-like zone containing many enzymesand nutrient-carrier proteins.

The crystal violet-iodine complex is

GRAMS STAIN

peptidoglycan layer when the cells aretreated with a solvent.

Gram-negative cells are decolourised by thealcohol or acetone treatment, but are thenstained with safranin so they appear pink


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GRAMS STAIN

Thus, the essential difference between Gram-

positive and Gram-negative cells is theirability to retain the crystal violet-iodinecomplex when treated with a solvent.

ram-pos ve ac er a ave a re a ve y c wacomposed of many layers of the polymerpeptidoglycan (sometimes termed murein).

The thickness of this wall blocks the escapeof the crystal violet-iodine complex when thecells are washed with alcohol or acetone.


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GRAMS STAIN

Gram-negative bacteria have only a thin

layer of peptidoglycan, surrounded by athin outer membrane composed oflipopolysaccharide (LPS).

The crystal v olet- od ne complex s eas lylost through the LPS and thin peptidoglycanlayer when the cells are treated with a solvent.


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GRAMS STAIN

+Positive

-Negative


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GRAMS STAIN

The cultures to be stained should be

YOUNG Incubated in broth or on a solid medium

until growth is just visible (no more than 12to 18 hours old if possible).

Old cultures of some gram-positivebacteria may appear Gram negative.

This is especially true for endospore-forming bacteria, such as species from thegenus Bacillus.


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GRAMS STAIN

When feasible, the cultures to be stained

should be grown on a sugar-freemedium.

Many organisms produce substantial amounts of

capsular or slime material in the presence ofcerta n car o y rates.

This may interfere with decolorization, and certainGram-negative organisms such as Klebsiella mayappear as a mixture of pink and purple cells.


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BACTERIAL CELL WALLS

Made mostly of a rigid macromolecule

called PEPTIDOGLYCAN.

Peptidoglycan is composed ofN-

acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined by

1,4-glycosidic bonds

Chains of NAM and NAG are cross-linkedby peptide chains (differ amongbacterial species).


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Peptide chains are made of aa of D

configuration.

The most common peptides are four aminoacid long: L- alanine, D- alanine, D-glutamic

acid and lysine or diaminopimelic acid (DAP). NAM and NAG molecules form a repeating

structure. The strength of the bacterial cellwall is proportional to the extent of cross-linkages.

Covalently bound to the thick peptidoglycanare teichoic acid


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ENDOSPORES

Endospores are dormant alternate life

forms produced by: the genus Baci l lus(obligate aerobes found

in the soil); and

e genus l o s t r i d i um o ga eanaerobes often found as normal flora ofgastrointestinal tract of animals); and

several other less common genera


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ENDOSPORES

Function:An endospore is not a

reproductive structure but rather a resistant,dormant survival form of the organism.

Endospores are quite resistant to high

temperatures (including boiling), mosts n ectants, ow energy ra at on, ry ng, etc.

The endospore can survive possibly thousands ofyears until a variety of environmental stimuli

trigger germination, allowing outgrowth of asingle vegetative bacterium


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ENDOSPORES

Formation of Endospores

Under conditions of starvation, especially the lack ofcarbon and nitrogen sources, a single endosporesforms within some of the bacteria.

This process is called sporulation :1. First the DNA replicates and a cytoplasmic

membrane septum forms at one end of the cellforming a forespore.

2. The remainder of the vegetative cell engulfs theforespore.


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ENDOSPORES

Formation of Endospores continued

3. Then there is synthesis of peptidoglycan in the spacebetween the two membranes surrounding the foresporeto form the first protective coat, the cortex.

4. Calcium dipocolinate is also incorporated into the formingendospore.

5. Aspore coat composed of a keratin-like protein thenforms around the cortex. Sometimes an outer membranecomposed of lipid and protein and called an exosporiumis also seen.

6. Finally, the remainder of the bacterium is degraded andthe endospore is released. Sporulation generally takesaround 15 hours.


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Formation of Endospores


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ENDOSPORE STRUCTURE

The completed endospore consists of multiple

layers of resistant coats including: a cortex

loosely cross-linked peptidoglycan

a spore coat g y cross- n e era n an ayers o spore-spec cproteins

and sometimes an exosporium thin covering made of protein and lipids

These layers surround components of thevegetative cell a nucleoid, some ribosomes, RNA and enzymes.


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ENDOSPORE

STRUCTURE

ENDOSPORE RESISTANCE


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ENDOSPORE RESISTANCE Due to A variety of factors:

Proteinaceous spore coat- confersresistance to lysozyme and harsh chemicals

Calcium-di icolinate abundant within theendospore, may stabilize and protect the

endospore's DNA.

Specialized DNA-binding proteins saturatethe endospore's DNA and protect it fromheat, drying, chemicals, and radiation.


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CELL MEMBRANE

Critical functions:

Separates contents of the cells from theirexternal environment.

Membranes allow for the

compartmentalization of the cell. Membrane-bound compartments keep certain reactive

compounds away from other parts of the cell whichmight be affected by them.

Also reactants that are located in a small space are farmore likely to come into contact and hence the reactioncan be dramatically increased.

Unit 4 Bacteria

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